May 24, 2025
Non-enzymatic passaging of hPSC
- Valeria Fernandez Vallone1,
- Lyn Healy2,
- Nathalie Lefort3,
- Katarzyna Ludwik1,
- Tamer Onder4,
- Lisa Pavinato5,6,
- Fatma Visal OKUR7,
- Harald Stachelscheid1
- 1Core Unit pluripotent Stem Cells and Organoids - Berlin Institute of Health @ Charite, Berlin, Germany;
- 2The Francis Crick Institute;
- 3Université de Paris, Imagine Institute, iPSC Core Facility, INSERM UMR U1163, F-75015 Paris, France.;
- 4Koc University;
- 5Institute of Oncology Research (IOR), Bellinzona Institutes of Science (BIOS+), Bellinzona, Switzerland;
- 6Faculty of Biomedical Sciences, Università della Svizzera Italiana, Lugano, Switzerland.;
- 7Hacettepe University, Center for Stem Cell Research and Development (PEDISTEM) and Hacettepe University Faculty of Medicine, Department of Pediatrics
- CorEuStem
Protocol Citation: Valeria Fernandez Vallone, Lyn Healy, Nathalie Lefort, Katarzyna Ludwik, Tamer Onder, Lisa Pavinato, Fatma Visal OKUR, Harald Stachelscheid 2025. Non-enzymatic passaging of hPSC. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo32k7v4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 20, 2023
Last Modified: May 24, 2025
Protocol Integer ID: 91188
Keywords: passaging, EDTA, hPSC, iPSC, non-enzymatic, clumps, split, hpsc this protocol, hpsc, using enzyme, free reagent, enzyme
Funders Acknowledgements:
COST Action CorEuStem
Grant ID: CA20140
Abstract
This protocol describes the procedure to passage hPSC culture using enzyme-free reagents.
Guidelines
In this protocol, hPSC passaging is done in aggregate/clumps format. The protocol is adaptable to different enzyme-free reagents described in Materials section and variable plate formats.
Materials
LABORATORY EQUIPMENT AND CONSUMABLES
Use sterile material
- 1/5/10 mL pipettes
- 15/50 mL conical tubes
- Cell culture treated plastic vessels of choice e.g. 24, 12 or 6-well plates, T25, T75 flasks, 10cm dishes
- 10/200/1000µL tips and micropipettes (optional)
- Cell scraper
- Aspirator pump with disposable pipette
- Sterile filter unit with 0.22 µm filter
- Microscope, if available Stereo Microscope
- Incubator at 37oC and 5% CO2 or under hypoxic conditions, 5% O2/ 5% CO2
- Class II Biosafety Cabinet
MEDIA AND REAGENTS
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
ReLeSR™ 100 mL
STEMCELL Technologies Inc.Catalog #5872
Versene SolutionThermo FisherCatalog #15040033
Gentle Cell Dissociation Reagent 100 mL
STEMCELL Technologies Inc.Catalog #7174
DPBS no calcium, no magnesiumInvitrogen - Thermo FisherCatalog #14190136
UltraPure Distilled Water Invitrogen - Thermo FisherCatalog #10977-015
hPSC culture conditions and survival factors choice depend on hPSC line and individual lab practices. For options refer to protocols:
Before start
If using EDTA as enzyme-free reagent, prepare EDTA 0.5 millimolar (mM) as follows:
- Dilute 1 to 1000 EDTA 0.5 Molarity (M) in distilled water; e.g. 5 mL EDTA 0.5 Molarity (M) in 995 mL distilled water.
- Filter sterile using 0.22 µm filter.
- Store at RT.
Preparation of destination vessel
5m
Prepare coated tissue culture vessels and culture media according to hPSC line requirements and desired format.
Aspirate and discard coating solution from wells of destination vessel.
Add hPSC maintenance media per destination vessel, refer to Table 1 for recommended volumes.
| A | B | C | D | E | |
| Culture Vessel | DPBS (mL) | non-enzymatic reagent (mL) | Media for hPSC harvesting (mL) | Media in destination vessel (mL) | |
| 12 WP (per well) | 0.5 | 0.5 | 1.5 | 1 | |
| 6 WP (per well) | 1 | 1 | 2.5 | 2 | |
| T25 | 2 | 2 | 5 | 7 | |
| 10 cm | 3 | 3 | 7.5 | 10 | |
| T75 | 5 | 5 | 10 | 15 |
Table 1. Recommended volumes according to vessel format
5m
Keep prepared destination vessel at 37 °C until usage.
hPSC non-enzymatic passaging
51m
Prepare required volume of the reagents and media according to the Table 1. Equilibrate the media to Room temperature .
Note
Culture media aliquot to be used can be warmed up at 37°C for 00:15:00 . However, to preserve recombinant proteins activity Room temperature equilibration is recommended.
30m
Aspirate and discard media from selected source vessel.
1m
Wash the well once using DPBS (no Calcium/no Magnesium).
2m
Add enzyme-free reagent, refer to Table 1 for recommended working volumes.
1m
Incubate the vessel at 37 °C monitoring hPSC detachment using a microscope. Refer to Table 2 for recommended incubation times.
Cells will be ready to harvest when colonies start to be loosen though still attached.
| A | B | C | D | E | |
| Reagent | EDTA 0.5mM | GDR | ReLeSR | Versene | |
| Time (min) | 2-3 | 2-3 | 5-7 | 2-5 |
Table 2. Recommended incubation times according to enzyme-free reagent.
Note
This incubation can also be done at Room temperature , recommendation at 37 °C is to avoid hPSC stress.
5m
Aspirate and discard enzyme-free reagent.
1m
Add fresh hPSC maintenance media to the wells, refer to Table 1 for recommended harvesting volumes.
2m
Pipette up and down using 5 mL serological pipette against the bottom of the well to dislodge cells from the culture surface.
Note
Repeated execution of this step leads to progressively smaller hPSC aggregates, which can increase cellular stress and promote cell death. Aim to maintain a balance that results in a uniform aggregate suspension - ideally with sizes ranging from approximately 50-200 µm while minimizing shear stress.
Note
The use of scrapers is generally not recommended. However, for hPSC cultures with strong adherence properties, employing a scraper may be preferable to prolonged incubation with enzyme-free dissociation reagents.
3m
Collect hPSC aggregates suspension using 5 mL pipette and directly distribute drop-wise the approximate volume for splitting ratios: 1/10, 1/15, 1/20 (or as desired) to the destination vessel/s.
Note
Alternatively, aggregates suspension can be transferred to a conical tube prior distribution to better assess suspension volume.
5m
Gently move destination vessels with freshly passed aggregates in cross shape to distribute them evenly.
1m
Incubate the vessels at 37 °C and at 5 % volume CO2.
After 24:00:00 perform full media change. For further hPSC culture refer to protocol Maintenance of hPSC and protocol Reference pictures of hPSC cultured in defined conditions